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Background: Current standard medications for the treatment of allergic inflammation consist primarily of glucocorticoids and anti-histamines, but adverse side effects or insufficient responsiveness by patient subpopulations illustrate the need for safe and novel alternatives. Thus, there is a demand to develop a porcine model that is able to mimic mast cell-mediated type I hypersensitivity. Previously, we found that escin, a pharmacologically active mix of triterpene saponins from horse chestnut extracts, exerts anti-allergic effects in murine models and merits further investigation as an anti-allergic therapeutic. Methods: We developed a new porcine model of allergic dermatitis based on a clinical prick test protocol. Histamine clearly provoked erythema and swelling at the prick site, whereas the mast cell-degranulating compound 48/80 even more pronounced caused wheal and flare reactions known from the human prick response. This model was used to test the anti-allergic efficacy of orally applied escin. Results: Oral pretreatment of animals with escin strongly inhibited the allergic skin response induced by compound 48/80 in a dose-dependent manner. Additional in vitro data from murine mast cells indicate an engagement of the glucocorticoid receptor pathway upon treatment with escin. Conclusions: This model provides a valuable and easy-to-set-up tool for preclinical studies of mast cell-inhibiting compounds. The successful implementation of this model supports the development of oral escin applications as a novel anti-allergic therapy.

Whereas stereochemical purity in drugs has become the standard for small molecules, stereoisomeric mixtures containing as many as a half million components persist in antisense oligonucleotide (ASO) therapeutics because it has been feasible neither to separate the individual stereoisomers, nor to synthesize stereochemically pure ASOs. Here we report the development of a scalable synthetic process that yields therapeutic ASOs having high stereochemical and chemical purity. Using this method, we synthesized rationally designed stereopure components of mipomersen, a drug comprising 524,288 stereoisomers. We demonstrate that phosphorothioate (PS) stereochemistry substantially affects the pharmacologic properties of ASOs. We report that Sp-configured PS linkages are stabilized relative to Rp, providing stereochemical protection from pharmacologic inactivation of the drug. Further, we elucidated a triplet stereochemical code in the stereopure ASOs, 3'-SpSpRp, that promotes target RNA cleavage by RNase H1 in vitro and provides a more durable response in mice than stereorandom ASOs.

Recent GWAS have identified SNPs at a human chromosom1 locus associated with coronary artery disease risk and LDL cholesterol levels. The SNPs are also associated with altered expression of hepatic sortilin-1 (SORT1), which encodes a protein thought to be involved in apoB trafficking and degradation. Here, we investigated the regulation of Sort1 expression in mouse models of obesity. Sort1 expression was markedly repressed in both genetic (ob/ob) and high-fat diet models of obesity; restoration of hepatic sortilin-1 levels resulted in reduced triglyceride and apoB secretion. Mouse models of obesity also exhibit increased hepatic activity of mammalian target of rapamycin complex 1 (mTORC1) and ER stress, and we found that administration of the mTOR inhibitor rapamycin to ob/ob mice reduced ER stress and increased hepatic sortilin-1 levels. Conversely, genetically increased hepatic mTORC1 activity was associated with repressed Sort1 and increased apoB secretion. Treating WT mice with the ER stressor tunicamycin led to marked repression of hepatic sortilin-1 expression, while administration of the chemical chaperone PBA to ob/ob mice led to amelioration of ER stress, increased sortilin-1 expression, and reduced apoB and triglyceride secretion. Moreover, the ER stress target Atf3 acted at the SORT1 promoter region as a transcriptional repressor, whereas knockdown of Atf3 mRNA in ob/ob mice led to increased hepatic sortilin-1 levels and decreased apoB and triglyceride secretion. Thus, in mouse models of obesity, induction of mTORC1 and ER stress led to repression of hepatic Sort1 and increased VLDL secretion via Atf3. This pathway may contribute to dyslipidemia in metabolic disease.

SummaryInsulin resistance leads to hypertriglyceridemia and hepatic steatosis and is associated with increased SREBP-1c, a transcription factor that activates fatty acid synthesis. Here, we show that steatosis in insulin-resistant ob/ob mice was abolished by deletion of Scap, an escort protein necessary for generating nuclear isoforms of all three SREBPs. Scap deletion reduced lipid synthesis and prevented fatty livers despite persistent obesity, hyperinsulinemia, and hyperglycemia. Scap deficiency also prevented steatosis in mice fed high-fat diets. Steatosis was also prevented when siRNAs were used to silence Scap in livers of sucrose-fed hamsters, a model of diet-induced steatosis and hypertriglyceridemia. This silencing reduced all three nuclear SREBPs, decreasing lipid biosynthesis and abolishing sucrose-induced hypertriglyceridemia. These results demonstrate that SREBP activation is essential for development of diabetic hepatic steatosis and carbohydrate-induced hypertriglyceridemia, but not insulin resistance. Inhibition of SREBP activation has therapeutic potential for treatment of hypertriglyceridemia and fatty liver disease.

Online supplemental material. Table S1 shows the expression of genes related to drug metabolism in XBP1-deficient liver. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20111298/DC1., The mammalian stress sensor IRE1α plays a central role in the unfolded protein, or endoplasmic reticulum (ER), stress response by activating its downstream transcription factor XBP1 via an unconventional splicing mechanism. IRE1α can also induce the degradation of a subset of mRNAs in a process termed regulated IRE1-dependent decay (RIDD). Although diverse mRNA species can be degraded by IRE1α in vitro, the pathophysiological functions of RIDD are only beginning to be explored. Acetaminophen (APAP) overdose is the most frequent cause of acute liver failure in young adults in the United States and is primarily caused by CYP1A2-, CYP2E1-, and CYP3A4-driven conversion of APAP into hepatotoxic metabolites. We demonstrate here that genetic ablation of XBP1 results in constitutive IRE1α activation in the liver, leading to RIDD of Cyp1a2 and Cyp2e1 mRNAs, reduced JNK activation, and protection of mice from APAP-induced hepatotoxicity. A pharmacological ER stress inducer that activated IRE1α suppressed the expression of Cyp1a2 and Cyp2e1 in WT, but not IRE1α-deficient mouse liver, indicating the essential role of IRE1α in the down-regulation of these mRNAs upon ER stress. Our study reveals an unexpected function of RIDD in drug metabolism., National Institutes of Health (U.S.) (grant AI32412), National Institutes of Health (U.S.) (grant DK082448), National Institutes of Health (U.S.) (grant DK089211), American Heart Association

Lipid nanoparticles (LNPs) have proven to be highly efficient carriers of short-interfering RNAs (siRNAs) to hepatocytes in vivo; however, the precise mechanism by which this efficient delivery occurs has yet to be elucidated. We found that apolipoprotein E (apoE), which plays a major role in the clearance and hepatocellular uptake of physiological lipoproteins, also acts as an endogenous targeting ligand for ionizable LNPs (iLNPs), but not cationic LNPs (cLNPs). The role of apoE was investigated using both in vitro studies employing recombinant apoE and in vivo studies in wild-type and apoE(-/-) mice. Receptor dependence was explored in vitro and in vivo using low-density lipoprotein receptor (LDLR(-/-))-deficient mice. As an alternative to endogenous apoE-based targeting, we developed a targeting approach using an exogenous ligand containing a multivalent N-acetylgalactosamine (GalNAc)-cluster, which binds with high affinity to the asialoglycoprotein receptor (ASGPR) expressed on hepatocytes. Both apoE-based endogenous and GalNAc-based exogenous targeting appear to be highly effective strategies for the delivery of iLNPs to liver.

Triethylene glycol dimethacrylate (TEGDMA) is a resin monomer which is released from polymerized dental composite materials. It induced apoptosis in various target cells or inhibition of LPS-induced cytokine production in cells of the immune system after prolonged exposure. In these tissues, mitogen-activated protein kinases (MAPK) regulate signal transduction pathways that support cell survival and cytokine synthesis. The time-dependent regulation of MAPK as well as their linkage to the induction of apoptosis and cytokine release under the influence of resin monomers is unknown. It was the aim of the present study to investigate the kinetics of the up- or down-regulation of the MAPK p38, JNK, and ERK1/2, the induction of apoptosis and cytokine release in RAW264.7 mouse macrophages and human pulp-derived cells. ERK1/2, p38 and JNK were differentially activated by phosphorylation in the presence of lipopolysaccharide (0.1 microg/ml; LPS), a known inducer of MAPK activity, and TEGDMA (3 mM) as detected by Western blotting. In macrophages, ERK1/2 was activated about 6-fold by LPS, while no activation was observed in the presence of TEGDMA after 15 and 30 min. A slight activation of p38 was detected in cell cultures after short exposure to TEGDMA (30 min), but activated JNK was identified after LPS stimulation only. After a long 24 h exposure period, ERK1/2 and p38 were strongly activated by LPS, a combination of LPS/TEGDMA, and TEGDMA alone (15-20-fold). In human pulp-derived cells, ERK1/2 was phosphorylated after exposure to TEGDMA up to 2 h, and sustained activation of ERK1/2 as well as p38 (12-15-fold) was detected after prolonged exposure for 24 h. The LPS-induced, time-related increase in the secretion of the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) as well as the anti-inflammatory IL-10 was instantaneously inhibited by TEGDMA in mouse macrophages. In parallel, the percentage of cells in macrophage cultures in the stage of apoptosis and necrosis increased with exposure period. Yet, in contrast to the inhibition of cytokine release, apoptosis and necrosis caused by LPS and TEGDMA was a late response in both mouse macrophages and human pulp-derived cells. From these data it appears as if MAPK activation, inhibition of cytokine release and the induction of apoptosis and necrosis by TEGDMA are tightly related. The direct causal correlation of these phenomena, however, requires further investigation.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates low density lipoprotein receptor (LDLR) protein levels and function. Loss of PCSK9 increases LDLR levels in liver and reduces plasma LDL cholesterol (LDLc), whereas excess PCSK9 activity decreases liver LDLR levels and increases plasma LDLc. Here, we have developed active, cross-species, small interfering RNAs (siRNAs) capable of targeting murine, rat, nonhuman primate (NHP), and human PCSK9. For in vivo studies, PCSK9 and control siRNAs were formulated in a lipidoid nanoparticle (LNP). Liver-specific siRNA silencing of PCSK9 in mice and rats reduced PCSK9 mRNA levels by 50–70%. The reduction in PCSK9 transcript was associated with up to a 60% reduction in plasma cholesterol concentrations. These effects were shown to be mediated by an RNAi mechanism, using 5′-RACE. In transgenic mice expressing human PCSK9, siRNAs silenced the human PCSK9 transcript by >70% and significantly reduced PCSK9 plasma protein levels. In NHP, a single dose of siRNA targeting PCSK9 resulted in a rapid, durable, and reversible lowering of plasma PCSK9, apolipoprotein B, and LDLc, without measurable effects on either HDL cholesterol (HDLc) or triglycerides (TGs). The effects of PCSK9 silencing lasted for 3 weeks after a single bolus i.v. administration. These results validate PCSK9 targeting with RNAi therapeutics as an approach to specifically lower LDLc, paving the way for the development of PCSK9-lowering agents as a future strategy for treatment of hypercholesterolemia.

Significant effort has been applied to discover and develop vehicles which can guide small interfering RNAs (siRNA) through the many barriers guarding the interior of target cells. While studies have demonstrated the potential of gene silencing in vivo, improvements in delivery efficacy are required to fulfill the broadest potential of RNA interference therapeutics. Through the combinatorial synthesis and screening of a different class of materials, a formulation has been identified that enables siRNA-directed liver gene silencing in mice at doses below 0.01 mg/kg. This formulation was also shown to specifically inhibit expression of five hepatic genes simultaneously, after a single injection. The potential of this formulation was further validated in nonhuman primates, where high levels of knockdown of the clinically relevant gene transthyretin was observed at doses as low as 0.03 mg/kg. To our knowledge, this formulation facilitates gene silencing at orders-of-magnitude lower doses than required by any previously described siRNA liver delivery system.